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Abstract:

The present invention relates to isolated polypeptides which comprise an
amino acid sequence consisting of a mutated functional Abl kinase domain,
said mutated functional kinase domain being resistant to inhibition of
its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof, to the use of such
polypeptides to screen for compounds which inhibit the tyrosine kinase
activity of such polypeptides, to nucleic acid molecules encoding such
polypeptides, to recombinant vectors and host cells comprising such
nucleic acid molecules and to the use of such nucleic acid molecules in
the production of such polypeptides for use in screening for compounds
which inhibit the tyrosine kinase activity of such polypeptides.

Claims:

1. A method of detecting STI571 resistance in a subject, comprising:
selecting a subject undergoing STI571 therapy; obtaining a sample from
the subject; detecting a mutation in the amino acid sequence of an Abl
kinase domain of a Bcr-Abl polypeptide in the sample obtained from the
subject compared to position 255 of the Abl kinase domain of the amino
acid sequence set forth as SEQ ID NO: 2, wherein the mutation confers
resistance to kinase inhibition by STI571, thereby detecting STI571
resistance in the subject.

2. The method of claim 1, wherein the method comprises detection of an A
to T point mutation that results in the substitution of valine for
glutamic acid at position 255 or a G to A point mutation that results in
the substitution of lysine for glutamic acid at position 255.

3. The method of claim 1, wherein detecting a mutation in the Abl kinase
domain of the Bcr-Abl polypeptide comprises: determining the sequence of
a nucleic acid encoding the Abl kinase domain of the Bcr-Abl polypeptide
in the sample obtained from the subject; and comparing the amino acid
sequence encoded by the nucleic acid with the amino acid sequence set
forth as SEQ ID NO: 2; wherein a difference in the amino acid sequence of
the Abl kinase domain of the Bcr-Abl polypeptide from the sample obtained
from the subject compared to position 255 of the Abl kinase domain of the
amino acid sequence set forth as SEQ ID NO: 2 detects an STI571 resistant
Abl kinase domain polypeptide.

8. The method of claim 1, wherein detecting a mutation in an Abl kinase
domain of the Bcr-Abl polypeptide comprises: determining the sequence of
an Abl kinase domain in a sample from the subject; and comparing the
amino acid sequence of the Abl kinase domain with position 255 of the Abl
kinase domain of the amino acid sequence set forth as SEQ ID NO: 2,
wherein a difference in the amino acid sequence of the Abl kinase domain
from the sample from position 255 of the Abl kinase domain of the amino
acid sequence set forth as SEQ ID NO: 2 detects the STI571 resistant Abl
kinase domain polypeptide.

9. The method of claim 8, wherein the substitution is a substitution of
valine for glutamic acid or lysine for glutamic acid at position 255.

10. The method of claim 8, wherein the subject has a tumor.

11. The method of claim 10, wherein the tumor is a leukemia.

12. A method of detecting a STI571 resistant mutation in a subject,
comprising: selecting a subject for possible treatment with STI571;
obtaining a sample from the subject; detecting a mutation in an amino
acid sequence of the Abl kinase domain of a Bcr-Abl polypeptide in the
sample obtained from the subject compared to position 255 of the Abl
kinase domain of the amino acid sequence set forth as SEQ ID NO: 2,
wherein the mutation confers resistance to kinase inhibition by STI571,
thereby detecting an STI571 resistant mutation in the subject.

13. The method of claim 12, wherein the substitution is a substitution of
valine for glutamic acid or lysine for glutamic acid at position 255.

14. The method of claim 12, wherein detecting a mutation in an Abl kinase
domain of the Bcr-Abl polypeptide, comprises: determining the sequence of
the Abl kinase domain in the sample from the subject; and comparing the
amino acid sequence of the Abl kinase domain in the sample with position
255 of Abl kinase domain of the amino acid sequence set forth as SEQ ID
NO: 2, wherein a difference in the amino acid sequence of the Abl kinase
domain from the sample from position 255 of the Abl kinase domain of the
amino acid sequence set forth as SEQ ID NO: 2 indicates the presence of
mutant Abl kinase domain polypeptide in the subject.

15. The method of claim 12, wherein the subject has a tumor.

16. The method of claim 15, wherein the tumor is a leukemia.

17. The method of claim 16, wherein the leukemia is myelogenous leukemia.

18. The method of claim 14, wherein the subject is undergoing STI571
therapy and the method is used to detect the presence of the STI571
resistant Abl kinase domain mutation prior to clinical relapse.

19. The method of claim 7, wherein the leukemia is myelogenous leukemia.

20. The method of claim 11, wherein the leukemia is myelogenous leukemia.

21. The method of claim 1, wherein detecting the mutation in the amino
acid sequence of the Abl kinase domain of the Bcr-Abl polypeptide in the
sample compared to position 255 of the Abl kinase domain of the amino
acid sequence set forth as SEQ ID NO: 2 detects STI571 resistance in the
subject prior to clinical relapse of the subject during STI571 therapy.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 12/803,962, filed, Jul. 9, 2010, which is a continuation of U.S.
patent application Ser. No. 12/337,322, filed Dec. 17, 2008, now
abandoned, which is a continuation of U.S. patent application Ser. No.
11/343,891, filed Jan. 31, 2006, now abandoned, which is a continuation
of U.S. patent application Ser. No. 10/263,480, filed Oct. 3, 2002, now
abandoned. U.S. patent application Ser. No. 10/263,480 claims the benefit
of U.S. Provisional Application No. 60/327,387, filed Oct. 5, 2001.

FIELD OF THE INVENTION

[0002] This invention relates to isolated polypeptides which comprise an
amino acid sequence consisting of a mutated functional Abl kinase domain,
said mutated functional kinase domain being resistant to inhibition of
its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof, to the use of such
polypeptides to screen for compounds which inhibit the tyrosine kinase
activity of such polypeptides, to nucleic add molecules encoding such
polypeptides, to recombinant vectors and host cells comprising such
nucleic acid molecules and to the use of such nucleic acid molecules in
the production of such polypeptides for use in screening for compounds
which inhibit the tyrosine kinase activity of such polypeptides.

[0004] It was now surprisingly found that mutations present in the kinase
domain of the Bcr-Abl gene of patients suffering from CML or Ph+ALL
account for the biological resistance of these patients towards STI571
treatment in that said mutations lead to resistance of the Bcr-Abl
tyrosine kinase towards inhibition by STI571.

[0005] These findings are extremely valuable in e.g. finding new compounds
or combinations of compounds which are capable to overcome resistance
towards treatment with STI571. Moreover, knowledge of such mutations is
also very useful in the diagnosis of Ph+ leukemias in that it allows e.g.
the detection of drug-resistant clones before clinical relapse of the
patient.

DEFINITIONS

[0006] Within the context of this disclosure the following expressions,
terms and abbreviations have the meanings as defined below:

[0008] In the expression "a mutated functional Abl kinase domain", the
term "functional" indicates that the respective kinase domain possesses
tyrosine kinase activity. Preferably, the kinase activity of the mutated
functional Abl kinase domain is in the range of that of the native human
Abl kinase domain.

[0009] In the expression "a mutated functional Abl kinase domain being
resistant to inhibition of its tyrosine kinase activity by STI571 or a
salt thereof", the term "resistant" means that STI571 inhibits the
respective mutated functional Abl kinase domain with an IC50 that is
higher than that of the native human Abl kinase domain, i.e. higher than
about 0.025 μM, preferably higher than about 0.15 μM, more
preferably higher than about 0.25 μM, most preferably higher than
about 5 μM.

[0010] In the expression "amino acid sequence of the native human Abl
kinase domain or an essentially similar sequence thereof", the part "or
an essentially similar sequence thereof" refers to the amino acid
sequence of the native human Abl kinase domain containing mutations,
including amino acid exchanges, amino add deletions and/or amino acid
additions, that are not essential for the functionality of the kinase and
its resistance to inhibition by STI571 or a salt thereof within the
meaning of the term "functional" and "resistant" as defined hereinabove.

[0011] The expression "replaced by another amino acid" refers to the
replacement of a certain natural amino acid by another natural amino
acid.

[0012] The names of the amino acids are either written out or the one
letter or three letter codes are used. Mutations are referred to by
accepted nomenclature, e.g. "Ala380Thr" or "380 Ala→Thr" both
indicating that alanine at position 380 is replaced by threonine.

[0015] Unless indicated otherwise, the number given for a certain amino
acid refers to the numbering of the amino acids in SEQ ID NO:2. In an
amino acid sequence that is essentially similar to the amino acid
sequence of the native human Abl kinase domain within the meaning as
defined above, the amino acids are numbered in accordance with the
numbering of the amino acids in SEQ ID NO:2.

[0016] The term "isolated" means that the material is removed from its
original environment (e.g., the natural environment if it is naturally
occurring).

[0017] A "host cell", refers to a prokaryotic or eukaryotic cell that
contains heterologous DNA that has been introduced into the cell by any
means, e.g., electroporation, calcium phosphate precipitation,
microinjection, transformation, viral infection, and the like.

[0019] In particular, the polypeptides of the present invention can be
produced by recombinant DNA technology using techniques well-known in the
art. Methods which are well known to those skilled in the art can be used
to construct expression vectors containing the sequences encoding the
polypeptides of the invention and appropriate
transcriptional/translational control signals. A variety of
host-expression vector systems can be utilized to express the
polypeptides of the invention.

(1). The invention relates to an isolated polypeptide which comprises a
mutated functional Abl kinase domain that is resistant to inhibition of
its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (2) The invention further
relates in particular to an isolated polypeptide which comprises a
mutated functional Abl kinase domain comprising the amino acid sequence
of the native human Abl kinase domain or an essentially similar sequence
thereof in which at least one amino acid is replaced by another amino
acid, said mutated functional Abl kinase domain being resistant to
inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (3) The invention
especially relates to an isolated polypeptide which comprises a mutated
functional Abl kinase domain comprising the amino acid sequence of the
native human Abl kinase domain or an essentially similar sequence thereof
in which at least one amino acid selected from Leu248, Glu255, Lys271,
Glu286, Met290, Thr315, Tyr320, Asn322, Glu373, His 375 and Ala380 is
replaced by another amino acid, said mutated functional Abl kinase domain
being resistant to inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (4) A preferred
embodiment of the invention relates to an isolated polypeptide which
comprises a mutated functional Abl kinase domain comprising the amino
acid sequence of the native human Abl kinase domain or an essentially
similar sequence thereof in which at least one amino acid selected from
Leu248, Glu255, Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375
and Ala380 is replaced by another amino acid, said mutated functional Abl
kinase domain being resistant to inhibition of its tyrosine kinase
activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (5) Another preferred
embodiment of the invention relates to an isolated polypeptide which
comprises a mutated functional Abl kinase domain comprising the amino
acid sequence of the native human Abl kinase domain or an essentially
similar sequence thereof in which at least one amino acid selected from
Leu248, Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375 and Ala380
is replaced by another amino acid, said mutated functional. Abl kinase
domain being resistant to inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (6) Another especially
preferred embodiment of the invention relates to an isolated polypeptide
which comprises a mutated functional Abl kinase domain comprising the
amino acid-sequence of the native human Abl kinase domain or an
essentially similar sequence thereof in which at least one amino acid
selected from Glu255, Thr315 and Ala380 is replaced by another amino
acid, said mutated functional Abl kinase domain being resistant to
inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (7) Another very
preferred embodiment of the invention relates to an isolated polypeptide
which comprises a mutated functional Abl kinase domain comprising the
amino acid sequence of the native human Abl kinase domain or an
essentially similar sequence thereof in which at least one amino acid
selected from Glu255 and Ala380 is replaced by another amino acid, said
mutated functional Abl kinase domain being resistant to inhibition of its
tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (8) Most preferably the
invention relates to an isolated polypeptide according to any one of the
preceding paragraphs (2)-(7), wherein in the amino acid sequence of the
native human Abl kinase domain or an essentially similar sequence thereof
a single amino acid is replaced by another amino acid. (9) The invention
relates very especially preferred to an isolated polypeptide which
comprises a mutated functional Abl kinase domain comprising the amino
acid sequence of the native human Abl kinase domain or an essentially
similar sequence thereof in which Glu255 is replaced by another amino
acid, said mutated functional Abl kinase domain being resistant to
inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (10) Most especially
preferred the invention relates to an isolated polypeptide which
comprises a mutated functional Abl kinase domain comprising the amino
acid sequence of the native human Abl kinase domain or an essentially
similar sequence thereof that contains at least one amino acid mutation
selected from Glu255Val, Glu255Lys, Thr315Val, Thr315Leu, Thr315Met,
Thr315Gln, Thr315Phe and Ala380Thr, said mutated functional Abl kinase
domain being resistant to inhibition of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (11) In a further very
preferred embodiment the invention relates to an isolated polypeptide
which comprises a mutated functional Abl kinase domain comprising the
amino acid sequence of the native human Abl kinase domain or an
essentially similar sequence thereof that contains at least one amino
acid mutation selected from Glu255Val, Thr315Val, Thr315Leu, Thr315Met,
Thr315Gln, Thr315Phe and Ala380Thr, said mutated functional Abl
kinase-domain being resistant to inhibition of its tyrosine kinase
activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)benzamide or a salt thereof. (12) In another especially
preferred embodiment the invention relates to an isolated polypeptide
which comprises a mutated functional Abl kinase domain comprising the
amino add sequence of the native human Abl kinase domain or an
essentially similar sequence thereof that contains at least one amino add
mutation selected from Thr315Leu, Thr315Met, Thr315Gln and Thr315Phe,
said mutated functional Abl kinase domain being resistant to inhibition
of its tyrosine kinase activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (13) Most preferably the
invention relates to an isolated polypeptide according to any one of the
preceding paragraphs (10)-(12), wherein the amino acid sequence of the
native human Abl kinase domain or an essentially similar sequence thereof
contains a single amino acid mutation. (14) Preferred above all the
invention relates to an isolated polypeptide which comprises a mutated
functional Abl kinase domain comprising the amino acid sequence of the
native human Abl kinase domain or an essentially similar sequence thereof
that contains the amino acid mutation Glu255Val, said mutated functional
Abl kinase domain being resistant to inhibition of its tyrosine kinase
activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof. (15) In a preferred
embodiment the invention relates to an isolated polypeptide according to
any one of the preceding paragraphs (2)-(14), wherein the amino acid
sequence of the native human Abl kinase domain consists of amino acids
229-500 of SEQ ID NO:2. (16) In another preferred embodiment the
invention relates to an isolated polypeptide according to any one of the
preceding paragraphs (2)-(15), said isolated polypeptide being a Bcr-Abl
tyrosine kinase. (17) In yet another preferred embodiment the invention
relates to the use of an isolated polypeptide of any one of the preceding
paragraphs (2) to (16) to screen for compounds which inhibit the tyrosine
kinase activity of said polypeptide. (18) The invention also relates to
an isolated nucleic acid molecule comprising a nucleotide sequence that
encodes a polypeptide according to any one of the preceding paragraphs
(2)-(16). (19) The invention further relates to the use of a nucleic acid
molecule of the preceding paragraph (18) in the production of a
polypeptide of any one of the preceding paragraphs (2) to (16) for use in
screening for compounds which inhibit the tyrosine kinase activity of
said polypeptide. (20) The invention also relates to a recombinant vector
comprising a nucleic acid molecule according to the preceding paragraph
(18). (21) The invention further relates especially to a recombinant
vector according to the preceding paragraph (20), which is a recombinant
expression vector. (22) The invention also relates to a host cell
comprising a recombinant vector according to the preceding paragraph (20)
or (21).

[0020] Preferably the invention relates to an isolated polypeptide which
comprises a mutated functional Abl kinase domain comprising the amino
acid sequence of the native human Abl kinase domain in which at least one
amino acid is replaced by another amino acid, said mutated functional Abl
kinase domain being resistant to inhibition of its tyrosine kinase
activity by
N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-pi-
perazin-1-ylmethyl)-benzamide or a salt thereof.

[0021] Most preferred are the mutations described herein, which are
present in patients who suffer from Philadelphia chromosome-positive
leukemia and are resistant against treatment with STI571.

[0022] A preferred salt of STI571 is the methane sulfonate salt described
in WO 99/03854.

[0023] Screening for compounds which inhibit the tyrosine kinase activity
of the polypeptides of the invention may be done for example by using an
isolated polypeptide of the invention in any in vitro tyrosine kinase
phosphorylation assay known in the art and determining the potential of a
compound to inhibit the tyrosine kinase activity of a polypeptide of the
invention in such an assay.

[0024] High-throughput screening assays known in the art may be used to
screen large compound libraries for compounds which inhibit the tyrosine
kinase activity of the polypeptides of the invention.

[0025] Besides the random screening of large compound libraries, the
polypeptides of the present invention may also be used in the following
screening approach: The 3-dimensional structure of a polypeptide of the
invention is determined by e.g. X-ray crystallography. The atomic
coordinates of a polypeptide of the invention are then used to design a
potential inhibitor. Said potential inhibitor is then synthesized and
tested for its ability to inhibit the tyrosine kinase activity of the
polypeptide of the invention in any in vitro tyrosine kinase
phosphorylation assay.

EXAMPLES

[0026] The following Examples serve to illustrate the invention without
limiting its scope.

Example 1

Methods:

Plasmids and Site Directed Mutagenesis:

[0027] The hybrid cDNA coding for HckAblSH1 was cloned by amplifying the
respective DNA fragments from pUCΔNdeI/XbaIHck [Warmuth M. et al.,
J. Biol. Chem. 272, 33260-70 (1997)] and pcDNA3bcr-abl. These fragments
were ligated blunt end to yield pUCΔNdeI/XbaIhckablSH1. Because of
its relatively small size when compared to Bcr-Abl or c-Abl, this
construct, hckAblSH1, allowed to introduce point mutations into the
kinase domain of Abl by a one step cloning procedure. Point mutations
were introduced into hckablSH1 using the QUICKCHANGE® site directed
mutagenesis protocol from Stratagene (La Jolla, Calif.). In order to
introduce point mutations into Bcr-Abl, a KpnI/Eco47III-subfragment of
Bcr-Abl containing the sequence coding for Bcr-Abl's kinase domain was
cloned into pUCΔNdeI/XbaI engineered by site directed mutagenesis
to contain an Eco47111 site in the polylinker. After introduction of
point mutations, this fragment was first recloned into pcDNA3abl.
Thereafter, the 5' part of abl up to the KpnI site was substituted by Bcr
coding sequences using a KpnI-fragment from pcDNA3bcr-abl. All mutations
were confirmed by sequencing. For expression in Cos7 and 32D cells cDNAs
were cloned into pApuro.

[0029] Cos7 cells were transfected using EFFECTEN® transfection reagent
as to the guidelines of the manufacturer (Quiagen, Hilden, Germany). 32D
cells were transfected by electroporation. Puromycin was used for
selection at a concentration of 1 μg/ml.

[0033] For assessing apoptosis induced by the various kinase inhibitors,
cells were incubated with the indicated concentrations of STI571 at a
density of 5×104 per ml. Apoptosis was routinely assessed by
measuring the binding of FITC-conjugated Annexin V to the membranes of
apoptosing cells. About 5×104 cells were taken at the
indicated time points and washed once in PBS. Thereafter, cells were
resuspended in 195 μl of Annexin V binding buffer and 5 μl of
Annexin V-FITC (Bender MedSystems Diagnostics, Vienna, Austria) were
added. Cells were mixed and incubated at room temperature for 10-20 min.
Afterwards, cells were pelleted again, washed once and resuspended in 190
μl of Annexin V binding buffer. 10 μl of a 20 μg/ml propidium
iodide stock solution were added and the ratio of Annexin V-positive to
negative cells was determined by FACS-analysis using a Coulter EPICS XL
4-color cytometer.

Results:

[0034] Mutations to either Valine (V), Leucine (L), Isoleucine (I),
Methionine (M), Glutamine (Q) or Phenylalanine (F) at position 315 and to
either Serine (S), Cysteine (C) or Threonine (T) at position 380 were
generated in a hybrid kinase, HckAblSH1, consisting of the SH2 and SH3
domain of Hck and the SH1/kinase domain of Abl. When expressed in Cos7
cells, these hybrid kinases and all mutants at position 315 and 380
showed a high spontaneous kinase activity, proving that these positions
are not critical for ATP binding (Table 1). In marked contrast, when
tested for inhibition by STI571, no inhibition was seen with up to 10
μM of compound for the mutants T315L, T315I, T315M, T315Q and T315F
(Table 1), whereas HckAblSH1 wild-type (wt) could be inhibited with
similar kinetics by STI571 as were found for Bcr-Abl (IC50 cellular
tyrosine phosphorylation (IC50CTP) approx. 0.5 μM). The mutants
T315V and A380T retained some partial sensitivity but IC50CTP values
were still higher than 10 μM. In contrast, the mutants A380S and A380C
displayed sensitivity to STI571, which was comparable to HckAblSH1 wt
(see Table 1 for summary).

All data based on inhibition of cellular tyrosine phosphorylation of
transiently transfected Cos7 cells determined by Western blot analysis
using the monoclonal α-phosphotyrosine antibody PY99. IC50 values
were determined using scion image software. Complete remission (CR) was
defined as no detectable reduction of cellular tyrosine phosphorylation
by 10 μM STI571. NS (normal sensitivity)=inhibition with similar
kinetics as HckAblSH1 wt.

[0035] Our data identify positions 315 and 380 as critical gatekeepers for
the binding pocket of STI571, which contribute to define the sensitivity
of individual protein kinases towards STI571. For example, the
STI571-insensitive receptor tyrosine kinase Flt-3, which has high
homology to the c-Kit and the PDGF-R kinases, has a phenylalanine at the
position homologous to T315, which would, based on our data, not be in
accordance with STI571 binding. In a similar way, the resistance of most
other kinases tested with STI571 could be explained.

[0036] In order to investigate whether and to what degree some of the
above described point mutations of the gatekeeper position T315 are able
to induce biological resistance towards STI571 we introduced into full
length Bcr-Abl the mutations T315V, T315L, T315I, T315M, T315Q and T315F.
When expressed in Cos7 cells all mutants displayed kinase activity close
to or similar to wild-type Bcr-Abl (Bcr-ABLwt). If tested for inhibition
by STI571, identical results were obtained as described for the
corresponding mutations in HckAblSH1 (Table 2). Similar to Scr-Ablwt,
expression of these mutants in 32D, an IL-3-dependent, hematopoietic cell
line of murine origin, gave rise to cell lines growing IL-3
independently. Exposure of 32 DBcr-Ablwt cells to 1 or 10 μM STI571
lead to a rapid stop of proliferation and induction of apoptotic cell
death in more than 90% of cells. On the contrary, if T315 mutant Bcr-Abl
kinases, for example T315I, were expressed the block in proliferation and
the induction of apoptosis caused by 1 μM STI571 were completely
abolished (Table 2) and the effects of STI571 seen at 10 μM were
reduced to levels found in control experiments using parental 32D cells
grown in the presence of IL-3. Phosphotyrosine blots of samples of cells
expressing either wt or mutant Bcr-Abl proteins confirmed that mutations
at position 315 completely abolished the effect of STI571 on Abl auto-
and substrate phosphorylation, with the exception of T315V which was
still to some degree inhibited by STI571 but displayed a similar
biological phenotype as the other mutants (Table 2). This suggests that
the reminder biological activity of STI571 at 10 μM was rather due to
cytotoxicity than to a reminder sensitivity of the mutants or
cross-reaction of STI571 with another tyrosine kinase. In summary, all
mutations lifted the IC50 for inhibition of proliferation (IC50IOP)
from 0.09 to approximately 7.5 μM and for inhibition of survival
(IC50IOS) from 0.5 to more than 10 μM (Table 2). Taken together,
these data show that mutations of "molecular gatekeeper" positions as
described above are able to confer complete biological resistance towards
STI571 in a cell culture model.

[0037] STI571 inhibits the Abl tyrosine kinase with an IC50 of 0.025
μM for purified Bcr-Abl and c-Abl but not the fms or the Src family
kinases. The mechanism of inhibition is through competitive inhibition of
ATP binding. To better understand the mechanism of specificity of the
tyrosine kinase inhibitor the Abl kinase was compared to a model of the
Lck kinase domain. This model predicts the following sites are critical
for STI571 association: L248, Y320, N322, E373, H375 and A380. Each of
these residues were changed to the corresponding residue in Src or fms
and IC50 values for STI571 with each mutant were determined. L248A
and H375L yielded kinase inactive mutants, Y320K, N322S, E373N and A380G
had IC50 values identical to wild type Abl. A380T, however,
demonstrated an IC50 of 0.34 μM suggesting that STI571 bound less
efficiently when a larger residue replaced the alanine. Recent
crystallization of the Abl kinase domain with a related inhibitor shows
that the configuration of the activation loop of the Abl kinase domain
differs significantly from that of the Src family kinases. This structure
identifies K271, E286, M290, T315, M318 and D381 as critical contacts of
STI571. All of these residues are conserved between Src and Abl. The last
two of these bind STI571 via their peptide backbone, thus mutants in
these residues cannot be created. The remainder of the residues were
mutated to residues lacking the potential for hydrogen bonding and
IC50 values were determined. K271R, E286L and M290A were kinase
inactive. T315V had an IC50 value of 0.35 μM, which is consistent
with the crystal structure of the Abl kinase domain which predicts that
the side chain of T315 forms a critical hydrogen bond with STI571.

Example 3

[0038] A group of 32 patients who are either refractory to treatment with
STI571 or who relapsed whilst being treated were investigated. The median
duration of therapy was 95 days; prior to STI571 treatment, two patients
were in chronic phase, nine in accelerated phase, 20 in myeloid and, and
one in lymphoid blast crisis of the disease. Reverse
transcriptase-polymerase chain reaction (RT-PCR) products specific for
the Bcr-Abl tyrosine kinase domain were sequenced

[0039] (Heminested RT-PCR was performed to amplify the sequence
specifically coding for the Bcr-Abl tyrosine kinase:

[0040] An acquired A→T point mutation at position 58802 (GeneBank
accession number U07563, locus HSABLGR3)--which results in a Glu255Val
substitution--was detected in one patient. Restriction analysis of cDNA
and genomic DNA (RT-PCR and genomic PCR were performed using primers A4+
TCACCACGCTCCATTATCCA, A4-CTTCCACACGCTCTCGTACA; Mnl I restriction digest
of PCR products; removal of an Mnl I restriction site as the result of
the point mutation A58802T) was used to confirm the presence of the
mutation and to track it during the course of treatment. Only wild-type
Abl sequence was present before the STI571 therapy. The patient was
treated with STI571 in late chronic phase, went into complete hematologic
remission, but progressed to blast crisis after five months. Reactivation
of Bcr-Abl was confirmed by Crk1 immunoblotting [K. Senechal, Mol. Cell.
Biol. 18, 5082 (1998)]. The relative proportion of phosphorylated Crk1
(reflecting active Bcr-Abl) was 49% before STI571 therapy, 24% at day 27,
28% at day 83, and 77% at the time of clinical resistance at day 166. The
biological significance of the Glu255Val change is determined by an Abl
autophosphorylation assay. STI571 inhibits wild-type Abl with an
IC50 of 0.025 μM. The mutation leads to a virtual insensitivity
to STI571, with an IC50 of >5 μM.

Example 4

[0041] The Bcr-Abl kinase domain from cells obtained from 12 CML and Ph+
acute leukemia patients who relapsed while receiving STI571 was
sequenced. A functional point-mutation in the kinase domain in one case
was identified. This was a G→A change that results in a
Glu→Lys substitution at position 255 of Abl.

Example 5

Patients and Sample Preparation:

[0042] Thirty bone marrow samples from 21 patients with Ph+ALL who were
enrolled into consecutive "Phase II study to determine the safety and
anti-leukemic effect of STI571 in adult patients with Ph+ acute
leukemias" were analyzed. According to the study protocol, these patients
had relapsed ALL or were refractory after at least 2 cycles of standard
chemotherapy. From all of the patients, samples were obtained before
beginning STI571 treatment: 13 of these samples were from individuals who
later were classified as good responders to STI571 (Nos. 1-13, sensitive,
S) including 12 patients with hematological complete remission (CR) and
one patient with partial remission (PR) but complete peripheral
hematological recovery (No. 1). Eight samples were collected from
individuals who later were found not to respond to STI571 (Nos. 14-21,
primarily resistant, R) including 6 patients without any hematological
response, one with cytoreduction in the bone marrow but persistent
peripheral leukemic cells (No. 20) and another with PR but incomplete
peripheral hematological recovery (No. 16). Matched bone marrow samples
from 9 patients (Nos. 1-5 and Nos. 14-17) were also obtained while they
were on treatment with STI571. Mononuclear cells were separated by
density gradient centrifugation through Ficoll-Hypaque (Biochrom, Berlin,
Germany). Total RNA was extracted using the acid
guanidium/phenol/chloroform method with minor modifications. [Puissant C.
and Houdebine L. M., Biotechniques 8, 148-149 (1990)]. Only samples with
leukemic blast cell infiltration of more than 80% were included into the
analysis.

[0044] Analysis of the sequence of the ATP binding site revealed a single
point mutation at nucleotide 1127 (GI6382056) changing a G to an A
resulting in a substitution at codon 255 of Lys (mutant) for a Glu
(wild-type). This mutation was found in 6 samples from patients after
they were treated with STI571 (Nos. 1, 2, 4, 5, 15, 16) but mutations
were not found in any other sample including the matched samples from the
patients before beginning treatment with STI571 (Table 3). The change was
verified by sequencing from both the sense and antisense directions. In
addition, one sample (No: 17) from a patient with an aberrant CALL had a
single point mutation at nucleotide 1308 changing a C to T resulting in a
substitution at codon 315 of isoleucine (mutant) for a threonine
(wild-type). This sample was unusual because the cells also expressed
CD33, a cell surface protein expressed on myeloid cells.

[0045] Our data strongly suggest that E255K developed during treatment
with STI571. Our analysis of matched samples found, that none of the
samples from untreated patients (including sensitive patients and those
with primary resistance) had this mutation. In contrast, six of 9 samples
(67%) from these patients undergoing treatment with STI571 had this
substitution at E255. The overall frequency of mutations in the ATP
binding site was 7 of 9 (78%) in our paired bone marrow samples from
patients undergoing therapy with STI571.